1. Field Of the Invention.
The present invention relates to centrifugation and more particularly to the execution of centrifugation in a controlled manner by simulation analysis.
2. Description of Related Art
Essentially, centrifugation is a process for separating particles suspended in a solution. In biological applications, the particles are usually macromolecules, cells, DNA fragments, etc. The device used for centrifugation is a centrifuge which includes a rotor that supports several containers (e.g. centrifuge tubes) of sample solution for rotation about a common axis. As the rotor spins in the centrifuge, centrifugal force is applied to each particle in the sample solution and each particle will sediment at a rate which is proportional to the centrifugal force applied. The viscosity of the sample solution and the physical properties of the particle also affect the sedimentation rate of each individual particle. The sedimentation speed of the particle is proportional in part to its shape and size (molecular weight), and to the ratio of the particle and solution densities.
One of the many methods of centrifugal separation is by density gradient centrifugation, which permits the complete separation of several or all of the components in a mixture according to their buoyant densities. The density gradient method involves a supporting column of "density gradient" fluid whose density increases toward the bottom of the tube. The density gradient fluid consists of a suitable low molecular weight solute dissolved in a solvent in which the sample particle mixture can be suspended.
In using the centrifuge to purify circular DNA plasmids, geneticists and molecular biologists frequently use an isopycnic separation technique. In this technique, both circular (desirable) and non-circular (non-desirable contaminant) DNA plasmids are saturated with ethidium bromide and then suspended in a concentrated solution of cesium chloride (CsCl). High speed centrifugation of the suspension results in the formation of a CsCl concentration gradient (and hence density gradient), and separation of the relatively dense circular DNA from the relatively light non-circular DNA/ethidium bromide complexes in the density gradient. Each particle will sediment or float toward a position in the centrifuge tube at which the gradient density is equal to its own buoyant density, and there it will remain in equilibrium. The isopycnic technique, therefore, separates particles into zones or bands on the basis of their buoyant density differences.
An important consideration in designing and executing such centrifugation separations is the need to prevent excessive sedimentation of the dissolved CsCl, which can lead to CsCl crystallization at the tube bottom and consequent excessive local rotor stress levels. In U.S. Pat. No. 5,171,206 (assigned to the assignee of the present invention and incorporated by reference herein), and also in "Computer Derived Rotor Speed Protocols for in situ Control of Local Solute Concentration during Centrifugation", Proceedings of the 112th Annual Meeting of the American Society of Mechanical Engineers, BED-Vol. 21, Bioprocess Engineering Symposium, Book. No. H00726-1991, pp. 9-13, a method of obtaining optimal centrifugal separation subject to the CsCl crystallization constraint is disclosed. Such method can be used in plasmid separations and allows a computer on board the centrifuge to calculate the solute distributions and the rotor speed vs. time protocol that absolutely maximizes rotor speed, subject to the constraint that CsCl crystal formation is forbidden. The method involves the numerical integration of the partial differential equation of sedimentation-diffusion (the Lamm equation) for three solutes (CsCl, circular DNA and non-circular DNA) while the simulated rotor speed is subject to the constraint that there be no change in the CsCl concentration at the tube bottom upon reaching saturation (i.e., the solubility limit). The aforementioned numerical integration gives, as outputs, the optimized rotor speed as a function of time, and the solute concentrations as a function of space and time. A characteristic of the aforementioned simulation method is that it has no natural simulation endpoint. Left to its own device, it will continue indefinitely to determine solute concentrations as functions of space and time, and rotor speed as a function of time, even though the concentrations and rotor speed asymptotically approach limits. For the optimization method to be practical, therefore, some other method needs to be invoked concurrently that periodically checks the status of the simulated run and tells the computer when the simulated DNA separation is complete, at least within specified limits.